Activation by hormone of adenylate cyclase is dependent on the presence of guanine nucleotides which act at a nucleotide regulatory site. The actions of GTP at the regulatory site are readily reversible, in contrast to the actions of analogs such as Gpp(NH)p and Gpp(CH2)p which produce persistent activation. We have found that the alpha-beta methylene analog, Gp(Ch1)pp, is also an effective activator of hepatic adenylate cyclase and exhibits characteristics similar to Gpp(NH)p. The stability of the alpha-beta methylene bond makes it highly unlikely that the so-called "persistent" activation results from covalent modification of the enzyme by guanine nucleotides, as has been suggested in the literatures. We have also shown that activation by these analogs is slowly reversible, which suggests that the persistence of activation results from tight binding of the analogs to the nucleotide regulatory site. The fact that all of the analogs which produce the slowly reversible activated state are stable to hydrolysis at the beta-gamma position suggests that the regulatory site contains a GTPase.